Nucleic acid probe technology has developed rapidly in recent years as researchers have discovered its value for detection of various diseases, organisms or genetic features which are present in very small quantities in a test sample. The use of probes is based upon the concept of complimentarily. For example, DNA is double-stranded, the strands bound to each other by hydrogen bonds between complementary nucleotides (also known as nucleotide pairs).
The DNA complex is normally stable, but the strands can be separated (or denatured) by conditions which disrupt the hydrogen bonding. The released single strands will reassociate only with another strand having a complementary sequence of nucleotides. This hybridization process can occur in solution or on a solid substrate. RNA is usually single-stranded. It can also hybridize with another strand or portion thereof which has a complementary sequence of nucleotides.
A target nucleic acid sequence of the DNA or RNA of a target organism or cell may be only a small portion of the total strand, so that it is very difficult to detect its presence using most known labeled probes. Much research has been carried out to overcome this problem including improvements in probe sensitivity and synthesis of nucleic acids.
A significant advance in the art is the process described in U.S. Pat. No. 4,683,202 (issued Jul. 28, 1987 to Mullis). Without going into extensive detail regarding that process, it is an amplification technique wherein primers are hybridized to nucleic acid templates in the presence of a polymerization agent (such as a polymerase) and four nucleotide triphosphates, and extension products are formed from the primers. These products are denatured and used as templates in a cycling reaction which amplifies the number and amount of existing nucleic acids to facilitate their subsequent detection. The amplification process of Mullis can be carried out cyclically as many times as desired to produce a larger quantity of detectable material from a small amount of target nucleic acid sequence.
In the process of U.S. Pat. No. 4,683,202, two primers are used for each strand of the target nucleic acid to be amplified. In the best case for amplification, the nucleic acid sequence to be amplified is completely complementary with the primer, at least near the 3' end of the target sequence. Thus, only one primer per strand is needed for effective amplification. It is known from the Mullis patent that where the target sequence is not entirely known, at least at the 3' end, a collection of primers can be used having all possible codon variations in order to have at least one primer which is completely complementary. Such a primer is said to have 100% "homology" with the end of the strand to be amplified.
This procedure may sometimes accomplish the amplification process desired, but it may be inefficient or ineffective in other instances. The preparation of the collection of random primers is wasteful and leads to the use of competitive non-extending primers. Moreover, when the uncertainty of the target nucleotide sequence is greater, the collection of primers needed is greatly enlarged.
Mismatches between target sequence and primers cannot be entirely avoided, particularly when the target sequence cannot be identified completely. In other instances, such as the detection of provital DNA from retroviruses, the target nucleic acid is highly variable, and complete identity cannot be maintained. With HIV-I, a variety of sequences in the genome produces a viable virus. Base substitutions are known to occur at random and frequent intervals over the entire genome. Thus, isolates are likely to have provital DNA which have different nucleic acid sequences.
Such mismatches will considerably reduce the efficiency of amplification by primers. In other words, mismatches lead to a slowing down of the amplification process because the kinetics of priming and primer extension have changed [see for example, an article by Tinoco, Jr., Proc. Nat. Acad. Sci.(USA), 85, 6252 (1988)]. In the worst case, no amplification will occur as the primer fails to attach to the target, or if it attaches, formation of an extension product is inhibited (that is, the primer "misfires").
It would be desirable to have an efficient means for amplifying nucleic acid sequences even if there are suspected mismatches between the target sequence and a known primer to that sequence.